Conjugate and Polysaccharide Vaccines

Conjugate and Polysaccharide Vaccines, Plenary Review

Meningococcal polysaccharide-protein conjugate vaccines DM Granoff Chiron Biocine, Emeryville, California, and Children’s Hospital Oakland Research Institute, Oakland, California Meningococcal polysaccharide vaccines are available for prevention of invasive diseases caused by Neisseria meningitidis, serogroups A, C, Y and W135. Protection correlates with the ability of vaccination to induce serum complement-mediated bactericidal antibody(1) In general, the antibody responses to these unconjugated polysaccharides are age-dependent: these vaccines are highly effective in adults (1), but they elicit negligible or incomplete and short-lived protection in infants and preschool children (1,2). Polysaccharide vaccines for prevention of disease caused by serogroup B meningococcal strains are not available. The group B polysaccharide is a poor immunogen at all ages, possibly because of immunologic tolerance induced in the host by the presence of cross-reactive polysialyated glycoproteins in fetal and adult tissues (4). Most polysaccharides appear to elicit antibody responses largely without the need for T-cell help (i.e., they are thymic-independent, or TI antigens). Conjugation of a polysaccharide to a protein carrier profoundly alters the immunologic properties of the polysaccharide, converting it from a TI to a thymic-dependent (TD) antigen. In the resulting TD conjugate, the immunogenicity of the polysaccharide is greatly enhanced, giving rise to IgG anticapsular antibodies and memory B cells. Experience with Haemophilus type b conjugate vaccines. Much has been learned about human immunity to polysaccharide-protein conjugate vaccines from studies of Haemophilus influenzae type b (Hib) vaccines. Compared to unconjugated Hib polysaccharide, the immunogenicity of the conjugated Hib polysaccharide in infants is greatly enhanced, and repeated injections elicit IgG booster responses, whereas such responses are not observed after repeated doses of unconjugated Hib polysaccharide (1). Serum antibody to the type b capsule confers protection against invasive Hib disease. In addition, Hib conjugate vaccination primes for long-term immunologic memory to the Hib polysaccharide, a property not elicited by vaccination with unconjugated Hib polysaccharide or even after recovery from Hib disease (1). The ability to develop memory B cells, which leads to rapid IgG anticapsular antibody responses upon encountering Hib organisms, may be an important alternative mechanism of protection against developing disease in vaccinated individuals who either have shown subprotective antibody responses to Hib conjugate vaccination (1), or whose serum antibody concentrations have declined to below the protective level (1). Hib conjugate vaccines are effective in two other important ways: first, the anticapsular antibody elicited by repeated injections of Hib conjugate vaccines undergoes affinity maturation (author’s unpublished data). The resulting higher avidity antibodies are more efficient at activating complement-mediated bacteriolysis or opsonization of Hib than lower avidity antibodies (1,2). Second, immunization with Hib conjugate vaccines not only protects the individual from developing invasive Hib disease by inducing protective immunity, but also

lowers the rate of nasopharyngeal colonization and transmission of Hib in the population (1,2). By this mechanism, Hib conjugate vaccination can have a better effect on decreasing the incidence of Hib disease in the population than would be predicted based on vaccine coverage (1). Experience with meningococcal polysaccharide-protein conjugate vaccines. Meningococcal oligosaccharide- and polysaccharide-protein conjugate vaccines have been prepared for prevention of diseases caused by serogroups A, B, and C organisms (1,2,3,4,5). To date, data from humans are limited to serogroup A and C meningococcal oligosaccharide-protein conjugate vaccines. A first-generation prototype vaccine was prepared at Chiron Biocine using meningococcal A and C oligosaccharides that are independently coupled to CRM197 carrier protein (a cross-reactive nontoxic mutant diphtheria toxin) (17). The conjugation method is based on selective end-reducing group activation of oligosaccharides and subsequent coupling to the protein through a six-carbon “spacer” molecule, adipic acid. In adults, the immunogenicity of the first-generation meningococcal A and C conjugate vaccine appeared to be similar to that of a control unconjugated meningococcal polysaccharide vaccine (1). A “second-generation” meningococcal C vaccine was prepared using similar chemistry except that very small oligomers (degree of polymerization [Dp] less than six monomers in length) were removed prior to conjugation (1). In phase I and II clinical trials in humans, both vaccines have been shown to be very safe in infants, toddlers and adults (1,2,3,4,17,18,19). Expanded clinical trials with the second-generation meningococcal C conjugate vaccine are currently in progress. Immunogenicity. Toddlers. In a study of US toddlers 18 to 23 months of age, conducted at the University of California, Los Angeles (UCLA), two doses of the Chiron Biocine meningococcal A and C conjugate vaccine given two months apart elicited 50- to >100-fold higher bactericidal antibody responses to both group A and group C strains, compared to those observed in control toddlers vaccinated with two doses of unconjugated meningococcal polysaccharide vaccine (Table 1) (21). Interestingly, the relative differences in immunogenicity between the conjugate and unconjugated vaccines in this study were much less striking when the antibody responses of the toddlers were measured by an ELISA, performed using a consensus protocol developed at the Centers For Disease Control (i.e., relative differences in antibody responses of 2-fold, instead of 50- to >100-fold, as determined by the bactericidal assay). These results suggest that the anticapsular antibody elicited by this conjugated meningococcal oligosaccharide vaccine is qualitatively different from that elicited by unconjugated meningococcal polysaccharide vaccine and, on a µg/ml basis, the conjugate-induced antibodies have a higher specific functional activity. Further, the ELISA is insensitive to these qualitative antibody differences, which may be important in protection against developing meningococcal disease. Table 1. Serum bactericidal antibody responses of US toddlers vaccinated with a meningococcal A and C oligosaccharide-CRM conjugate vaccine* Geometric Mean Bactericidal Titer (Reciprocal)

Meningococcal Bactericidal Antibody

Conjugate Vaccine (N = 42)

Polysaccharide Vaccine (N = 41)

Anti-A PrePost-2

8

7

756

38

5

4

319 8

11

Anti-C PrePost-2

*Toddlers were given two doses of either meningococcal A and C oligosaccharide-CRM197 conjugate vaccine, prepared at Chiron Biocine, or meningococcal polysaccharide vaccine (Menomune, Connaught Laboratories, Inc.). The injections were separated by two months. Complement-mediated bactericidal activity was measured in serum obtained immediately before dose one (Pre-) and one month after dose two (Post-2). Adapted from Lieberman J, et al. JAMA 1996;275:1499–1503. (21) The “second-generation” Chiron Biocine meningococcal C polysaccharide-protein conjugate vaccine, in which small saccharide oligomers are removed prior to conjugation, was recently evaluated in toddlers 12 to 23 months of age in a multicenter Canadian study (19). This vaccine also elicited much higher serum bactericidal antibody responses after one or two injections than those observed in sera of control toddlers vaccinated with unconjugated polysaccharide vaccine. Infants. The safety and immunogenicity of Chiron Biocine meningococcal conjugate vaccines also have been investigated in infants less than six months of age. The first study was performed with the combined A and C conjugate vaccine in Gambian infants immunized at 2, 3 and 4 months of age, or 2 and 6 months, or 6 months of age (20). The anticapsular antibody responses of the infants to the A component of the conjugate vaccine were of similar magnitude to those of a control group receiving an unconjugated meningococcal A and C polysaccharide vaccine. In contrast, the conjugate vaccine elicited two- to four-fold higher anticapsular antibody responses to meningococcal C polysaccharide than those observed in infants given the unconjugated polysaccharide vaccine. However, the elevated anti-C antibody concentrations in the conjugate group began to decline within three months after vaccination. In the Gambian study, only ELISA antibody responses were reported. Based on the experience in the UCLA toddler study described above (21), the ELISA results may have underestimated the relative effectiveness of the conjugate vaccine as compared to unconjugated polysaccharide, had the responses been assessed by serum bactericidal antibody. In a study in the UK, 58 infants were vaccinated with the combined Chiron Biocine meningococcal A and C conjugate vaccine at 2, 3 and 4 months of age (23). To date, antibody responses to the group A vaccine have not been evaluated. However, the infants showed excellent anti-meningococcal C bactericidal responses after the first and second injections (geometric mean bactericidal titer of 1:100 after one injection, and >1:1000 after two injections). There was no further increase in titer after the third injection. There are two other reports of the use of meningococcal C conjugate vaccines in infants. In one study, the “second-generation” Chiron Biocine meningococcal C polysaccharide-protein conjugate vaccine was given to UK infants at 2, 3 and 4 months of age (22). Preliminary results were limited to assays of sera obtained prior to vaccination and one month after the third injection. The infants showed 25-fold increases in anticapsular antibody concentrations, as measured by ELISA, and >50-fold increases in bactericidal titers. In the other study, a meningococcal serogroup C oligosaccharide-CRM197 conjugate vaccine was prepared by Lederle Praxis Biologics and administered to US infants at 2, 4 and 6 months of age (16). This conjugate vaccine appeared to be well tolerated and elicited significant increases in serum anticapsular antibody concentration after two or three injections, as assessed by ELISA. Induction of immunologic memory. In the study of Gambian infants given the Chiron Biocine first-generation combined meningococcal A and C vaccine, serum antibody concentrations to both polysaccharides had begun to decline within three months after conjugate vaccination (20). An important question, therefore, is whether or not the conjugate vaccination induced memory B cells that might allow the infants to respond rapidly with an increase in serum anticapsular antibody concentration upon encountering group A or C meningococci. To examine this question, participants in this study were re-vaccinated at 18 to 24 months of age with an unconjugated meningococcal polysaccharide vaccine (1). Prior to the booster injection, the serum antibody concentrations to the A or C polysaccharides in the groups previously given the conjugate vaccine were not significantly different from those of toddlers of similar age who had not been previously vaccinated. However, as shown in Table 2, the toddlers primed with meningococcal conjugate vaccine at 2 and 6 months of age developed much higher antimeningococcal C bactericidal titers after the polysaccharide booster vaccination than did toddlers of similar age immunized for the first time with unconjugated meningococcal polysaccharide vaccine. In contrast, the Gambian toddlers who had been primed with meningococcal unconjugated polysaccharide vaccine at 3 and 5 months of age showed significantly lower anti-C bactericidal responses to the booster injection than the control toddlers vaccinated for the first time (Table 2). This result confirms previous data suggesting that immunization at an early age with unconjugated meningococcal group C polysaccharide vaccine induces immunologic tolerance and impairs the ability to respond to a subsequent immunization with unconjugated meningococcal polysaccharide vaccine (1). The clinical importance of this finding is unknown. However, immunologic tolerance to meningococcal C polysaccharide could enhance susceptibility to developing invasive meningococcal disease by impairing the ability of the child to develop a protective serum anticapsular antibody response upon encountering the organism. Although the Gambian toddlers who had been given conjugate vaccine as infants were primed for memory antibody responses to meningococcal C polysaccharide, similar priming was not observed to the group A polysaccharide: that is, the magnitude of the anti-A antibody response to the booster injection of unconjugated polysaccharide vaccine in the conjugate-primed group was not significantly higher than that observed in Gambian toddlers vaccinated with unconjugated meningococcal polysaccharide for the first time (Table 2). Further, in contrast to meningococcal C, evidence of immunologic tolerance to meningococcal A polysaccharide was

not observed in the toddlers previously given the unconjugated polysaccharide vaccine at 3 and 5 months of age (Table 2). Indeed, the toddlers previously vaccinated with unconjugated polysaccharide vaccine appear to have shown secondary antibody responses to the serogroup A polysaccharide. These data confirm the results of many previous studies indicating that group A and group C meningococcal unconjugated polysaccharide vaccines have very different immunologic properties (summarized in reference 4, Frasch, 1995): specifically, meningococcal A polysaccharide vaccines appear to be both immunogenic and protective in early infancy (2), and may even prime for secondary antibody responses to a subsequent injection (25). Meningococcal C polysaccharide vaccine shows none of these properties. The immunologic basis for these differences remains unknown. Recently, toddlers who participated in the UCLA study of the Chiron Biocine meningococcal A and C conjugate vaccine were also given a booster injection of unconjugated meningococcal polysaccharide vaccine approximately one year later (1). The group that had been primed with the conjugate vaccine showed evidence of induction of memory B cells to both the serogroup A and C polysaccharides, as evidenced by very high serum bactericidal booster antibody responses. These results are in contrast to those of the Gambian infants given this conjugate vaccine in whom evidence of induction of memory B cells was limited to the serogroup C polysaccharide, and not the serogroup A polysaccharide (see above) (24). Further studies of immunologic priming induced by meningococcal A conjugate vaccines at different ages and in different populations are needed to clarify this discrepancy.

Table 2. Effect of priming with meningococcal A and C oligosaccharide-CRM conjugate vaccine on serum bactericidal antibody responses of Gambian toddlers boosted with unconjugated meningococcal polysaccharide vaccine* Geometric Mean Bactericidal Titer (Reciprocal) 10–14 Days Post-Booster Priming Vaccine Meningococcal Bactericidal Antibody

None (N = 34)

Polysacchari de Vaccine (N = 17)

Conjugate Vaccine (N = 15)

Anti-A

338

178 3

549

Anti-C

239

26

439 0

*Toddlers in the Gambia were boosted with a dose of unconjugated meningococcal polysaccharide vaccine (Menpovax A plus C, Biocine, Siena, Italy) at a mean age of 19.7 months. The subjects had either been previously vaccinated at 3 and 6 months of age with unconjugated meningococcal polysaccharide, or at 2 and 6 months of age with meningococcal A and C oligosaccharide-CRM conjugate vaccine (Twumasi, et al.) (20). A group of control unprimed toddlers from the same study area were vaccinated for the first time. Data shown are bactericidal titers measured in sera obtained 10 to 14 days after the booster vaccination (adapted from Leach A, et al., J Infect Dis 1996, in press) (24). Meningococcal B polysaccharide-protein conjugate vaccines. Investigational polysaccharideprotein conjugate vaccines also have been prepared for prevention of disease caused by serogroup B organisms (1,2,3,15). In general these conjugates are much less immunogenic than those prepared with serogroup A or C polysaccharides. However, one promising immunogenic meningococcal B vaccine candidate is a conjugate in which the polysaccharide component has been modified by substitution of N-propionyl groups for N-acetyl groups (NPr-meningococcal B polysaccharide) (28). To date, there are no published data from trials in humans with this type of conjugate. However, it will be a difficult task to prove that such vaccines are safe in humans because, in mice, data from our laboratory suggest that NPr-meningococcal B polysaccharideprotein conjugate vaccines induce anti-NPr-meningococcal B polysaccharide antibodies that cross-react with native NAc-meningococcal B polysaccharide and also appear to have autoantibody activity (Bartoloni A, et al., unpublished data). Whether or not the use of alternative substitutions, such as N-butanoyl, will truly permit an antibody response that is functional, entirely pathogen-specific, and not cross-reactive with host antigens, remains to be ascertained (1).

Conclusions and the future. Meningococcal A and C conjugate vaccines hold enormous promise for providing solid long-term protection to infant age groups that are currently poorly protected by licensed unconjugated meningococcal polysaccharide vaccines. Further, “thirdgeneration” conjugate vaccines are under development in which the acquisition of immunity is accelerated and immunogenicity is enhanced by administration of the conjugate vaccine with novel adjuvants (1). The use of adjuvants suitable for humans holds promise for decreasing the number of doses of conjugate vaccine required for induction of immunity in infants. This approach also may be useful for enhancing or maintaining immunogenicity of future multicomponent conjugate vaccines. For prevention of group B meningococcal disease, adjuvanted polysaccharide-protein conjugate vaccines also may prove to be immunogenic and protective. However, alternative approaches may be needed to avoid the safety concerns of inducing anticapsular antibodies with autoantibody reactivity. Acknowledgement Supported, in part, by a grant (V23/181/169) from the World Health Organization Global Programme for Vaccines. References 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Artenstein MS, Gold R, Zimmerly JG, Wyle FA, Schneider H, Harkins C. Prevention of meningococcal disease by group C polysaccharide vaccine. N Engl J Med 1970; 282:417–20. Reingold AL, Broome CV, Hightower AW, et al. Age-specific differences in duration of clinical protection after vaccination with meningococcal polysaccharide A vaccine. Lancet 1985; 2:114–8. Frasch CE. Meningococcal vaccines: past, present and future. In: Cartwright K, ed. Meningococcal disease. New York: John Wiley & Sons, 1995:245–83. Daum RS, Granoff DM. Lessons from the evaluation of immunogenicity. In: Ellis RW, Granoff DM, eds. Development and Clinical Uses of Haemophilus b Conjugate Vaccines. New York: Marcel Dekker, Inc., 1994:291–312. Weinberg GA, Einhorn MS, Lenoir AA, Granoff PD, Granoff DM. Immunologic priming to capsular polysaccharide in infants immunized with Haemophilus influenzae type b polysaccharide - Neisseria meningitidis outer membrane protein conjugate vaccine. J Pediatr 1987; 111:22–27. Granoff DM, Anderson EL, Osterholm MT, et al. Differences in the immunogenicity of three Haemophilus influenzae type b conjugate vaccines in infants. J Pediatr 1992; 121:187–194. Granoff DM, Holmes SJ, Osterholm MT, et al. Induction of immunologic memory in infants primed with Haemophilus influenzae type b conjugate vaccines. J Infect Dis 1993; 168:663–671. Schlesinger Y, Granoff DM, the Vaccine Study Group. Avidity and bactericidal activity of antibody elicited by different Haemophilus influenzae type b conjugate vaccines. JAMA 1992; 267:1489–1494. Lucas AH, Granoff DM. Functional differences in idiotypically defined IgG1 anti-polysaccharide antibodies elicited by vaccination with Haemophilus influenzae type b polysaccharide-protein conjugates. J Immunol 1995; 154:4195–4202. Takala AK, Eskola J, Leinonen M, et al. Reduction of oropharyngeal carriage of Haemophilus influenzae type b (Hib) in children immunized with an Hib conjugate vaccine. J Infect Dis 1991; 164:982–6. Murphy TV, Pastor P, Medley F, Osterholm MT, Granoff DM. Decreased Haemophilus colonization in children vaccinated with Haemophilus influenzae type b conjugate vaccine. J Pediatr 1993; 122:517–523. Murphy TV, White KE, Pastor P, et al. Declining incidence of Haemophilus influenzae type b disease since introduction of vaccination. JAMA 1993; 269:246–248. Beuvery EC, Miedema F, van Delft R, Haverkamp K. Preparation and immunochemical characterization of meningococcal serogroup C polysaccharide-tetanus toxoid conjugates as a new generation of vaccines. Infect Immun 1983; 40:39–45. Beuvery EC, Kaaden A, Kanhai V, Leussink AB. Physiochemical and immunological characterization of meningococcal serogroup A polysaccharide-tetanus toxoid conjugates prepared by two methods. Vaccine 1993; 1:31–6. Jennings HJ, Lugowski C. Immunochemistry of serogroups A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates. J Immunol 1981; 127:1011–18. Rennels MB, Edwards KM, Keyserling HL, et al. Immunogenicity and safety of conjugate meningococcal group C vaccine in infants. Abstract 1083, Pediatr Res 1996; 39:183A. Costantino P, Viti S, Podda A, Velmonte MA, Nencioni L, Rappuoli R. Development and phase 1 clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine 1992; 10:691–8. Anderson EL, Bowers T, Mink CM, et al. Safety and immunogenicity of meningococcal A and C polysaccharide conjugate vaccine in adults. Infect Immun 1994; 62:3391–3395.

 19. MacDonald NE, Halperin S, Law BJ, et al. Biocine meningococcal C (MenC) conjugate vaccine elicits high titers of serum bactericidal activity in toddlers. SPR abstract, Pediatr Research, May 1996 20. Twumasi Jr. PA, , Kumah S, Leach A, et al. A trial of a group A plus group C meningococcal polysaccharide-protein conjugate vaccine in African infants. J Infect Dis 1995; 171:632–8. 21. Lieberman JM, Chiu SS, Wong VK, et al. Safety and immunogenicity of a serogroups A/C Neisseria meningitidis oligosaccharide-protein conjugate vaccine in young children. JAMA 1996; 275:1499–1503. 22. Shackley FM, Heath PT, Flamank C, et al. Immunogenicity and reactogenicity of a group C meningococcal conjugate vaccine in British children (abstract). Annual meeting of The Infectious Diseases Society of America, Oct. 1996. 23. Fairley CK, Begg N, Borrow R, Fox AJ, Jones DM, Cartwright K. Conjugate meningococcal serogroup A and C vaccine: reactogenicity and immunogenicity in U.K. infants. Unpublished data. 24. Leach A, Twumasi PA, Kumah S, et al. Induction of immunological memory in Gambian children by immunization in infancy with a group A plus group C meningococcal polysaccharide and protein-conjugate vaccine. J Infect Dis 1996; in press. 25. Gold R, Lepow ML, Goldschneider I, Gotschlich EC. Immune response of human infants to polysaccharide vaccines of groups A and C Neisseria meningitidis. J Infect Dis 1977; 136(suppl):S31–5. 26. Lieberman JM, Wong VK, Partridge S, et al. Bivalent A/C meningococcal conjugate vaccine in toddlers: persistence of antibodies and response to a polysaccharide vaccine booster (abstract). 10th International Pathogenic Neisseria Conference. Baltimore, 1996. 27. Devi SJN, Robbins JB, Schneerson R. Antibodies to poly[(2->8)-α-N-acetylneuraminic acid] and poly[(2->9)-α-Nacetylneuraminic acid] are elicited by immunization of mice with Escherichia coli K92 conjugates: potential vaccines for groups B and C meningococci and E. coli K1. Proc Natl Acad Sci 1991 ; 88:7175–9. 28. Jennings HJ, Roy R, Gamian A. Induction of meningococcal group B polysaccharide-specific IgG antibodies in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine. J Immunol 1986; 137:1708–13. 29. Bartonoli A, Norelli F, Ceccarini C, et al. Immunogenicity of meningococcal B polysaccharide conjugated to tetanus toxoid or CRM197 via adipic acid dihydrazide. Vaccine 1995; 13:463–70. 30. Jennings HJ. Improved meningococcal polysaccharide conjugate vaccine. 1995; European patent specification, publication number: 0 504 202 B1. 31. Granoff DM, McHugh YE, Van Nest GA, Raff H. MF59 adjuvant enhances anticapsular antibody responses of infant primates vaccinated with meningococcal C (MenC) and Haemophilus type b (Hib) oligosaccharide (OS)-protein conjugate vaccines (abstract). Interscience Conference on Antimicrobial Agents and Chemotherapy. New Orleans: American Society for Microbiology, 1996.

Conjugate and Polysaccharide Vaccines

Protective epitope of N-propionylated group B meningococcal polysaccharide. HJ Jennings, R Pon, M Lussier and Q-L Yang Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON, Canada, K1A 0R6. Neisseria meningitidis is a human pathogen of worldwide significance. Although group B N. meningitidis is the most pathogenic serogroup its capsular polysaccharide is precluded from the current polysaccharide vaccine due to its poor immunogenicity (1). Furthermore this problem, which is attributed to structural mimicry between the group B meningococcal polysaccharide (GBMP) and human tissue antigens (2), cannot be satisfactorily overcome by coupling the GBMP to protein carriers (3). Currently there is no vaccine against group B meningococcal meningitis and most efforts to develop an effective vaccine have focused on sub-capsular components such as outer membrane proteins and lipopolysaccharides. Problems have been encountered in the development of these vaccines not the least of which is the intrinsic antigenic diversity exhibited by the components (4). Because the GBMP is a conserved antigenic structure on group B meningococci, a polysaccharide-based vaccine would be the vaccine of choice, provided one could overcome its poor immunogenicity. One simple way to achieve this goal is to use a synthetic vaccine composed of the Npropionylated (NPr) form of the GBMP, which when conjugated to tetanus toxoid (TT), induces in mice high titer antibodies that are bactericidal for all group B meningococci (5). The NPrGBMP-TT produced two distinct populations of antibodies, one of which (minor population) cross-reacted with the GBMP. Of significance to the development of a vaccine was that the major population of antibodies did not cross-react with the GBMP and yet contained all the bactericidal activity towards group B meningococci (6). In addition the induction of GBMP cross-reactive antibodies could also be reduced by adjuvant manipulation. Thus the NPr-GBMP must mimic a unique epitope on group B meningococci (6). In order to further define this epitope a series of mAb's of the IgG isotype were produced in BalbC mice using an (NeuPr)~35-TT conjugate vaccine. Most of the mAb's which were only minimally cross-reactive with the GBMP, recognized an extended helical form of the NPr GBMP. However, unlike GBMP-specific antibodies, which only recognize extended helical epitopes on the GBMP (2), a few were able to recognize short (random coil) segments of the NPr GBMP. Because of the paucity of clones specific for these short epitopes, additional mAb's with this specificity were generated using an (NeuPr)4-TT conjugate. Two important conclusions can be drawn from the properties of the above mAb's. The first is that while antibodies of the IgG1 isotype are not bactericidal, they confer good passive protection in mice challenged with live group B meningococci.The second is that regardless of isotype only mAb's specific for the extended helical form of the NPr GBMP are protective as defined by either passive protection experiments or bactericidal activity. Therefore one can draw the intriguing conclusion that whereas the serologically distinct extended helical epitopes of both the GBMP and the NPr GBMP co-exist in the capsular layer of group B meningococci and E. coli K1, only the former are present in purified α2-8-polysialic acid.

The presence of extended NPr GBMP-specific epitopes in the capsular layer of the above organisms was substantiated by electron microscopy, using a mAb with this specificity as the binding antibody.In addition using this technique, it was demonstrated that a mAb specific for short NPr GBMP epitopes did not bind to either organism, which is consistent with the lack of protection provided by mAb's with this specificity. References 1. Wyle FA, Artenstein MS, Brandt BL, et al. Immunologic response of man to group B meningococcal polysaccharide vaccines. J Infect Dis 1972; 126:514-22. 2. Häyrinen J, Jennings H, Raff HV. Antibodies to polysialic acid and its N-propyl derivative: binding properties and interaction with human embryonal brain glycopeptides. J Infect Dis 1995; 171:1481-90. 3. Jennings HJ, Lugowski C. Immunochemistry of group A , B and C meningococcal polysaccharide-tetanus toxoid conjugates. J Immunol 1982; 127:1011-18. 4. Frasch CE, Zollinger ND, Poolman JT. Serotype antigens of Neisseria meningitidis and a proposed scheme for designation of serotypes. Rev Infect Dis 1985; 7:504-10. 5. Jennings HJ, Roy R, Gamian A. Induction of meningococcal group B polysaccharidespecific IgG antibodies in mice by using an N-propionylated B polysaccharide -tetanus toxoid conjugate vaccine. J Immunol 1986; 137: 1708-13. 6. Jennings HJ, Gamian A, Ashton FE. N-propionylated group B meningococcal polysaccharide mimics a unique epitope on group B Neisseria meningitidis. J Exp Med 1987; 165: 1207-11.

Conjugate and Polysaccharide Vaccines

Preclinical evaluation of a combination vaccine against groups A, B, and C meningococci in both mice and nonhuman primates JY Tai, F Michon, PC Fusco, and MS Blake. North American Vaccine, Inc., Bethesda, Maryland, USA. Neisseria meningitidis is the major cause of bacterial meningitis worldwide. Meningococci of serogroups A, B, and C are responsible for approximately 90% of all the reported cases (1, 2). A combination polysaccharide-protein conjugate of each serogroup (A, B, and C) is being developed as a vaccine candidate against meningococcal infections. For group B, the polysaccharide was chemically modified at the C-5 position of the sialic acid residue wherein the N-acetyl groups are replaced with N-propionyl groups to alter the immune tolerance and provide greater immunogenicity (3, 4). The chemically modified B polysaccharide, as well as the native A and C polysaccharides, were coupled to the carrier protein, a recombinant class 3 porin (rPorB) of group B meningococci (5), by reductive animation (3). The rPorB was chosen as the carrier protein due to its ability to significantly increase the bactericidal activity towards the group B polysaccharide when conjugated (6, manuscript submitted for publication). The groups A, B, and C conjugates were evaluated individually and in combination in both mice and nonhuman primates (African green monkeys). Immune responses were assessed in terms of polysaccharide-specific IgG (by ELISA) and antibody-dependent complement-mediated bactericidal activity. In mice, the combination vaccine is highly immunogenic, eliciting high levels of polysaccharidespecific IgG and bactericidal activity against all three components. Booster effects were also clearly demonstrated after subsequent injections for all components indicating that T-dependency was achieved. No significant interference in immunological responses was observed for the trivalent vaccine formulation when compared with the monovalent vaccine controls. The responses in nonhuman primates for individual and combination vaccines are being evaluated, but initial results have shown significant bactericidal activity against all 3 serogroups after only one injection of the trivalent formulation. References 1. Riedo FX, Plikaytis BD, and Broome CV. Epidemiology and prevention of meningococcal disease. Pediatr Infect Dis J 1995; 14:643-657. 2. Hart CA and Rogers TR. Meningococcal disease. J Med Microbiol 1993; 39:3-25. 3. Jennings HJ, Roy R, and Gamian A. Induction of meningococcal group B polysaccharide-specific IgG antibodies in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine. J Immunol 1986; 137:1708-1713.

4. Jennings HJ, Gamian A, and Ashton FE. N-propionylated group B meningococcal polysaccharide mimics epitope on group B Neisseria meningitidis. J Exp Med 1987; 165:1207-1211. 5. Qi HL, Tai JY and Blake MS. Expression of large amounts of neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers. Infect Immun 1994; 62:2432-2439. 6. Tai JY, Michon F, and Fusco PC. Antibody-dependent, complement-mediated bactericidal activity elicited by group B meningococcal conjugate vaccines in mice and nonhuman primates. Abstract G3, American Society for Microbiology, 35th ICAAC 1995 (San Francisco, CA), Washington, D.C.

Conjugate and Polysaccharide Vaccines

Peptide mimic-induced primary human antibody response to the capsular polysaccharide of Neisseria meningitidis serogroup C MAJ Westerink , WA Hutchins , PK Holder , PL Pais , LL Gheesling , GM Carlone 1

1

2

2

2

2

Medical College of Ohio, Toledo, Ohio 43699 and Centers for Disease Control, Atlanta Georgia 30333.

1

2

Recent trials with a conjugated meningococcal vaccine preparation have failed to show enhanced immunogenicity over the conventional capsular polysaccharide vaccine (1). An alternate approach to convert the T-independent meningococcal vaccine into a T-dependent vaccine is through the use of an anti-idiotypic mimic of the native antigen. We have developed an antiidiotype-based peptide mimic of the capsular polysaccharide of Neisseria meningitidis serogroup C (MCPS). Immunization with this peptide complexed to proteosomes results in a protective anti-MCPS antibody response in Balb/c mice (2). Study of the human immune system has been hampered by the lack of experimental models to generate a primary immune response to Tindependent or T-dependent antigens. Mosier et al. (3) demonstrated mutant severe combined immunodeficient (SCID) mice could be engrafted with functional human peripheral blood lymphocytes (hu-PBL). A major limitation of the hu-PBL-SCID model has been the failure to demonstrate a primary human immune response (4-8). We hypothesized that the lack of consistent human primary immune response may be attributed to the lack of human cytokines resulting in impaired differentiation and maturation of human lymphoid cells in reconstituted SCID mice. We have developed a reliable system of inducing a human primary antibody response in the reconstituted hu-PBL SCID mouse model (9). This study was undertaken to define the optimal dose and configuration of MCPS peptide mimics. Three healthy volunteers were leukopheresed. Fifty five SCID mice/volunteer were reconstituted with 108 human lymphocytes and immunized with 10 µg MCPS; 10, 25, 50, or 100 µg of the P3 peptide (CARIYYRYDGFAY) complexed to proteosomes; 25 or 50 µg of 3xP3 peptide (IYYRYDIYYRYDIYYRYD) complexed to proteosomes; 25 or 50 µg of 3xYPY peptide (IYYPYDIYYPYDIYYPYD) complexed to proteosomes. The human anti-MCPS response was measured by ELISA. Functional activity was determined by bactericidal assay. The results of these studies showed that immunization with 50µg of P3 peptide complex or 25 µg of 3xP3 peptide complex resulted in the highest human anti-MCPS antibody titer (21.5 and 21.4 µg/ml respectively). Immunization with the 3xYPY peptide resulted in 10-15 µg/ml human antiMCPS antibody. Immunization with MCPS (one dose) results in 0.26 µg/ml anti-MCPS at 4 weeks. All antisera with an anti-MCPS titer exceeding 1 µg/ml proved to be functional in bactericidal assay. These data indicate that an anti-Id based peptide mimic of MCPS induces a protective, T-dependent antibody response in humans. References 1. Twumasi PA Jr., Kumah S, Leach A, et al. A trial of a group A plus group C meningococcal polysaccharide-protein conjugate vaccine in African infants. J Infect Dis 1995; 171:632-8.

2. Westerink MAJ, Giardina PC, Apicella MA, Kieber-Emmons T. Peptide mimicry of the meningococcal group C capsular polysaccharide. Proc Natl Acad Sci USA 1995; 92:4021-5. 3. Mosier DE, Gulizia RJ, Baird SM, Wilson DB. Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature 1988;335:256-259. 4. Mazingue C, Cottrez F, Auriault C, Cesbron JY, Capron A. Obtention of a human primary humoral response against schistosome protective antigens in severe combined immunodeficiency mice after the transfer of human peripheral blood mononuclear cells. Eur J Immunol 1991;21:1763-1766. 5. Markham R, Donnenberg B, Donnenberg AD. Effect of donor and recipient immunization protocols on primary and secondary human antibody responses in SCID mice reconstituted with human peripheral blood mononuclear cells. Infect Immun 1992;60:2305-2308. 6. Abedi MR, Christensson B, Islam KB, Hammarstram L, Smith CIE. Immunoglobulin production in severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood mononuclear cells. Eur J Immunol 1992;22:823-828. 7. Duchosal MA, Eming SA, Fischer P, et al. Immunization of HuPBL/SCID mice and the rescue of human monoclonal Fab fragments through combinatorial libraries. Nature 1992;335:258-262. 8. Nonoyama S, Smith FO, Ochs HD. Specific antibody production to a recall or a neoantigen by SCID mice reconstituted with human peripheral blood lymphocytes. J Immunol 1993;151:3894-3901. 9. Westerink MAJ, Adkins AR, Hutchins WA, et al. Primary human immune response to Neisseria meningitidis serogroup C in IL-12-treated hu-PBL-SCID mice. 1996. Submitted.

Conjugate and Polysaccharide Vaccines, Poster 69

Murine monoclonal antibodies to an N-propionylated meningococcal B polysaccharide exhibit heterogeneity with respect to cross-reactivity with N-acetylated meningococcal B polysaccharide and autoreactivity to host polysialyated glycoproteins A Bartoloni, S Ricci, E Gallo, D Rosa, N Ravenscroft, V Guarnieri, R Seid, A Shan, W Usinger, YE McHugh and DM Granoff Chiron Biocine, Siena, Italy and Emeryville, CA, and Children’s Hospital Oakland Research Institute, Oakland CA. The poor immunogenicity of the Neisseria meningitidis group B (MenB) polysaccharide capsule (PS), a homopolymer of α 2-8 sialic acid, represents a major challenge in developing an effective polysaccharide-based vaccine to prevent MenB disease. The poor immunogenicity of MenB PS has been attributed to immunologic tolerance induced from exposure to host polysialyated glycoproteins (e.g., neural cell adhesion molecules, termed N-CAMs). Substitution of Npropionyl (NPr) for N-acetyl (NAc) groups on the MenB PS, and conjugation of the resulting NPr MenB PS to a protein carrier, has been reported to result in a conjugate vaccine that is immunogenic in experimental animals and capable of eliciting protective antibodies that activate complement-mediated bactericidal activity (1). However, little is known about the crossreactivity of anti-NPr MenB PS antibodies with NAc MenB PS, or autoreactivity of these antibodies. To address these questions, we raised a panel of 28 murine anti-NPr MenB PS antibodies. After partial purification of the Mabs from tissue culture supernatants by ammonium sulfate fractionation and exhaustive dialysis, the Mabs were characterized for isotype, crossreactivity with NAc MenB PS by ELISA, and autoreactivity with a neuroblastoma cell line (CHP-134), which has been reported to express long chain α 2-8 linked polysialic acid (2). Of the 28 Mabs , one was IgM and the remaining 27 were IgG (three IgG1, three IgG2a, thirteen IgG2b and eight IgG3). Fourteen of the 28 antibodies (50%) cross-reacted with NAc MenB PS as demonstrated by direct binding to NAc MenB PS in a solid phase ELISA format. The specificity of this cross-reactivity was confirmed by inhibition of binding with soluble NAc MenB PS. The remaining 14 Mabs showed no cross-reactivity with NAc MenB PS when tested by ELISA at antibody concentrations up to 25 µg/ml. In preliminary studies, complementmediated bactericidal activity was detected among Mabs that cross-reacted with NAc MenB PS, and those that did not. However, the Mabs that cross-reacted with NAc MenB PS appeared to have the highest bactericidal activity (i.e., lowest concentrations needed to activate 50% killing [BC50]). Analysis of autoreactivity of the Mabs was performed using the CHP-134 human neuroblastoma (NB) cell line with and without neuraminidase (sialidase) treatment as a specificity control. Binding to this cell line was detected with several of the Mabs. Alternative approaches for measuring autoantibody activity are being used to confirm the pattern of reactivity. In conclusion, the murine anti-NPr MenB PS monoclonal antibodies described here are heterogeneous with respect to cross-reactivity with NAc MenB PS, their ability to bind to the NB cell line, and their ability to elicit complement-mediated bactericidal activity. Within the panel of Mabs there are examples of anti-NPr MenB PS Mabs that are bactericidal but do not cross-react with NAc MenB PS and do not show binding to the NB cell line. Presumably such antibodies could protect the host against the pathogen and pose no risk of eliciting autoimmune

disease. However, many of the anti-NPr MenB PS antibodies cross-reacted with native NAc MenB PS and also showed strong binding to the NB cell line. Although there is no evidence that the ability of an antibody to bind to host tissue will necessarily result in autoimmune disease, it will be a difficult task to prove that a conjugate vaccine that elicits such antibodies is safe to use in humans. References 1. Jennings HJ, Roy R, Gamain A. Induction of meningococcal group B polysaccharidespecific IgG antibodies in mice using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine. J Immunol 1986; 137:1708-1713 2. Livingston BD, Jacobs JL, Glick MC, Troy FA. Extended polysialic acid chains (n>55) from human neuroblastoma cells. J Biol Chem 1988; 263:9443-9448.

Conjugate and Polysaccharide Vaccines, Poster 70

Immunogenicity of a meningococcal serogroup A and C conjugate vaccine in UK infants. R Borrow , CK Fairley , N Begg , AJ Fox , DM Jones , and KAV Cartwright 1

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Public Health Laboratory, Withington Hospital, Nell Lane, Manchester, M20 2LR, UK. PHLS CDSC, 61 Colindale Avenue, London, NW9 5EQ, UK. Public Health Laboratory, Gloucester Royal Hospital, Great Western Road, Gloucester, GL1 3NN, UK. 1

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Currently approximately 1500 cases of meningococcal infections are notified each year in England and Wales with serogroup C disease accounting for about one third of infections (1). Serogroup C vaccines have been developed from capsular polysaccharide but, unconjugated, these vaccines do not protect those under two years of age (2). Similar experience with the Haemophilus influenzae type b (Hib) native polysaccharide led to the development of the Hib conjugate vaccines, which have demonstrated enhanced immunogenicity in young infants and the capacity to induce immunological memory (3). Initial trials addressing the safety and immunogenicity in adults of a serogroup A and C conjugate vaccine demonstrated significant rises in antibody levels to both A and C polysaccharide and bactericidal antibody titers to serogroup C meningococci (4). The most important role for an effective meningococcal conjugate vaccine is the protection of infants and children. Studies in infants in the Gambia with a serogroup A and C polysaccharideconjugate vaccine have demonstrated high levels of serogroup C antibodies although the bactericidal activities of these antibodies were not measured (5). In this study the immunogenicity of a serogroup A and C meningococcal polysaccharide-CRM197 conjugate vaccine was evaluated in 58 infants who received three doses at two, three and four months of age. Sera were tested for antibodies to the serogroup A and C capsular polysaccharide by enzyme-linked immunosorbent assay (ELISA) and bactericidal assays, against two serogroup C strains, using standardized protocols (6,7). The pre-vaccination total immunoglobulin geometric mean titers (GMT) to anti-A and C polysaccharide antibodies were respectively 2.8 and 0.6 µg/ml rising to 21.5 and 38.5 µg/ml one month after the third dose and falling to 3.1 and 2.2 µg/ml by 14 months of age. Pre-vaccination serum bactericidal titers against two serogroup C meningococci were